Various methods of introducing genes into the genome of plants are known (Halford N G, Shewry P R, Br Med Bull 2000; 56(1):62-73). The aim is the preparation of plants having advantageous, novel properties, for example to increase agricultural productivity, for improving the quality of foodstuffs or for producing particular chemicals or pharmaceuticals (Dunwell J M, J Exp Bot. 2000; 51 Spec No:487-96).
Moreover, the natural defence mechanisms of the plant, for example against pathogens, are inadequate. The introduction of foreign genes from plants, animals or microbial sources may enhance the defence, for example. Examples are the protection against insects feeding on tobacco by expression of the Bacillus thuringiensis endotoxin under the control of the 35 S CaMV promoter (Vaeck et al., Nature 1987, 328, 33-37) or the protection of tobacco against fungal infection by expression of a chitinase from beans under the control of the 35SCaMV promoter (Broglie et al., Science 1991, 254, 1194-1197).
It is furthermore possible to achieve resistance to herbicides by introducing foreign genes, thereby optimizing the cultivation conditions and reducing crop losses (Ott K H et al., J Mol Biol. 1996; 263(2):359-368).
The quality of the products may also be improved. Thus it is possible, for example, to increase the shelf life and storability of crop products by inactivating particular maturation genes. This has been demonstrated, for example, by inactivating polygalacturonase in tomatoes (Hamilton A J et al., Curr Top Microbiol Immunol 1995; 197: 77-89).
It is furthermore possible, by introducing further genes, advantageously metabolic genes, into plants, to enable particular products and by-products of naturally occurring metabolic processes to be utilized for a wide range of industries, including the feed, food, cosmetics and pharmaceutical industries. These molecules which are collectively referred to as “fine chemicals” include, for example, vitamins, amino acids, carbohydrates or lipids and fatty acids, one exemplary class of which are the polyunsaturated fatty acids (PUFAs). Polyunsaturated fatty acids are added, for example, to children's food in order to increase the nutritional value of these foods. PUFAs have, for example, a positive effect on the cholesterol level in the blood of humans and are therefore useful for protection against heart disease. Fatty acids and triglycerides have a multiplicity of applications in the food industry, animal nutrition, cosmetics and in the pharmaceutical sector.
A basic prerequisite for transgenic expression of particular genes in plants is the provision of plant-specific promoters. Various plant promoters are known. It is possible to distinguish between constitutive promoters which enable expression in various parts of a plant, which is only slightly restricted in terms of location and time, and specific promoters which allow expression only in particular parts or cells of a plant (e.g. root, seeds, pollen, leaves, etc.) or only at particular times during development. Constitutive promoters are advantageously used for expressing “selection markers”. Selection markers (e.g. antibiotic or herbicidal resistance genes) permit filtering of the transformation event out of the multiplicity of untransformed but otherwise identical individual plants.
In all cases, it is necessary to control specifically expression of the genes to be expressed, depending on the function of said genes. Any expressed genes in any organisms have a promoter region 5′ of the coding sequence. This region is responsible for the start of transcription itself and for regulating transcription. Said regulation is carried out usually by transcription factors binding to regulatory sequences within the promoter region. Promoters are usually freely portable within a species, i.e. it is possible to use a promoter of one gene in order to control transcription of another gene. This control of the new gene is then usually identical to controlling the original gene from which the promoter is derived. Thus it is possible to control expression of any gene in a known manner, using a known promoter whose regulation is known. This generally no longer applies, as soon as said promoter is used in other species. Thus, for example, promoters from the bacterium Streptomyces are recognized in the bacterium E. coli only poorly, if at all. The same applies to promoters of animal or plant origin which cannot readily be used reciprocally or in microorganisms.
Constitutive promoters active in plants have been described relatively rarely up to now. Promoters to be mentioned are the Agrobacterium tumefaciens TR double promoter, the promoters of the vacuolar ATPase subunits or the promoter of a proline-rich wheat protein (WO 91/13991) and also the Ppcl promoter Mesembryanthemum crystallinum (Cushman et al. (1993) Plant Mol Biol 21:561-566).
The constitutive promoters which are currently the predominantly used promoters in plants are almost exclusively of viral or bacterial origin, for example from Agrobacterium. In detail, these are the nopaline synthase (nos) promoter (Shaw et al. (1984) Nucleic Acids Res. 12(20):7831-7846), the mannopine synthase (mas) promoter (Comai et al. (1990) Plant Mol Biol 15 (3):373-381) and the octopine synthase (ocs) promoter (Leisner and Gelvin (1988) Proc Natl Acad Sci USA 85(5):2553-2557) from Agrobacterium tumefaciens and the CaMV35S promoter from cauliflower mosaic virus (U.S. Pat. No. 5,352,605). The latter is the most frequently used promoter in expression systems with ubiquitous and continuous expression (Odell et al.(1985) Nature 313:810-812; Battraw and Hall (1990) Plant Mol Biol 15:527-538; Benfey et al. (1990) EMBO J 9(69):1677-1684; U.S. Pat. No. 5,612,472). However, the CaMV 35S promoter which is frequently applied as constitutive promoter exhibits variations in its activity in different plants and in different tissues of the same plant (Atanassova et al. (1998) Plant Mol Biol 37:275-85; Battraw and Hall (1990) Plant Mol Biol 15:527-538; Holtorf et al. (1995) Plant Mol Biol 29:637-646; Jefferson et al. (1987) EMBO J 6:3901-3907). A further disadvantage of the 35S promoter is, for example, a change of it in transgene expression in the case of an infection with cauliflower mosaic virus and its typical pathogenic variants. Thus, plants expressing the BAR gene (Bialaphos resistance gene, alanylalanylphosphinothricine) under the control of the 35S promoter are no longer resistant after infection with the virus which typically occurs in nature (Al-Kaff et al. (2000) Nature Biotechnology 18:995-99).
From the range of viral promoters, the sugarcane bacilliform badnavirus (ScBV) which imparts an expression pattern similar to that of CamV has been described as an alternative to the CaMV 35S promoter (Schenk et al. (1999) Plant Mol Biol 39(6):1221-1230). The activity of the ScBV promoter was analyzed in transient expression analyses using various dicotyledonous plants, including Nicotiana tabacum and N. benthamiana, sunflower and oilseed rape, and monocotyledonous plants, here in the form of banana, corn and millet. In the transient analyses in corn, the ScBV promoter-mediated expression level was comparable to that of the ubiquitin promoter from corn (see below). Furthermore, the ScBV promoter-mediated rate of expression was assayed in transgenic banana and tobacco plants and displayed in both plant species essentially constitutive expression.
Common promoters for expressing selection markers in plants are especially the nos promoter, or else the mas promoter and ocs promoter, all of which have been isolated from Agrobacterium strains.
The use of viral sequences is often met with great reservations on the part of the consumer. These doubts are fed not least by studies which question the safety of the 35S CaMV promoter, owing to a possible horizontal gene transfer due to a recombination hot spot (Ho M W et al. (1999) Microbial Ecology in Health and Disease 11:194-197; Cummins J et al. (2000) Nature Biotechnology 18:363). It is therefore an aim of future biotechnological studies on plants to replace viral genetic elements by plant regulatory elements in order to keep as closely as possible to the plant system.
Owing to the prevailing doubts with regard to viral promoters, there are extensive efforts to replace said promoters by plant promoters. However, a promoter of plant origin, which is comparable to the viral elements, has not been described as yet.
What has been described, is a plant ubiquitin promoter from Arabidopsis thaliana (Callis et al. (1990) J Biol Chem 265:12486-12493; Holtorf S et al. (1995) Plant Mol Biol 29:637-747). However, some studies revealed that the Arabidopsis ubiquitin promoter is unsuitable for expressing selection marker genes.
Christensen et al. have described further promoters, namely the two corn ubiquitin promoters Ubi-1 and Ubi-2, which exhibit heat inducibility, in addition to constitutive basic expression (U.S. Pat. Nos. 5,510,474; 6,020,190 and 6,054,574). The expression pattern of the two promoters Ubi-1 and Ubi-2 from corn is described in Plant. Mol. Biol., (1992), 18(4):675-689. While the Ubi-1 promoter has good expression activity in corn and other monocotyledonous plants, it exhibits in dicotyledonous tobacco plants only 10% of the activity which had been achieved in comparable experiments using the viral 35S promoter. The corn Ubi-1 promoter is thus suitable for overexpression of genes in monocotyledonous plant systems. In addition, it is sufficiently strong in order to mediate a herbicidal resistance via expression of selection markers [Christensen and Quail (1996) Transgenic Res 5(3):213-218]. However, the Ubi-1 promoter proved unsuitable for dicotyledonous expression systems.
WO01/18220 describes a ubiquitin regulatory system which lacks the heatshock elements, i.e. it is no longer heat-inducible. This regulatory system was developed, starting from the corn Ubi-promoter system, by removing the heat-inducible elements.
Ubiquitins are omnipresent proteins which have been found in all eukaryotes analyzed thus far. Thus, Kawalleck et al. [Plant Molecular Biology, 21, 1993: 673-684] describe two parsley (Petroselinum crispum) ubiquitins, ubi4-1 and ubi4-2. The promoter of ubi4-2 has been isolated. It was not possible to demonstrate any heat inducibility of ubi4-1 and ubi4-2 under the conditions studied by Kawalleck et al.
A comparison of the organ specificity and strength of various constitutive promoters was carried out by Holtorf (Holtorf et al. (1995) Plant Mol Biol 29(4):637-646) on the basis of stably transformed Arabidopsis plants. The study comprised, inter alia, the CaMV35S promoter, the leaf-specific thionine promoter from barley and the Arabidopsis ubiquitin promoter (UBQ1). The CaMV35S promoter exhibited the highest rate of expression. On the basis of using an additional translational enhancer (TMV omega element), it was possible to increase the rate of expression of the promoter by a factor of two to three with unchanged organ specificity. The leaf-specific thionine promoter from barley was inactive in the majority of transformed lines, while the UBQ1 promoter from Arabidopsis resulted in medium rates of expression.
McElroy and colleagues reported a construct for transforming monocotyledonous plants, which is based on the rice actin 1 (Act1) promoter (McElroy et al. (1991) Mol Gen Genet 231:150-1609). Overall, it was concluded from the afore-described studies that the Act1promoter-based expression vectors are suitable for controlling a sufficiently strong and constitutive expression of foreign DNA in transformed cells of monocotyledonous plants.
Another constitutive promoter which has been described is the promoter of an S-adenosyl-L-methionine synthetase (WO 00/37662). A disadvantage here is especially a dependence of the strength of expression on the methionine concentration.
WO 99/31258 describes chimeric constitutive plant promoters which are composed of various elements of various promoters with complementary expression patterns so that the combination of individual tissue specificities additively results in a constitutive expression pattern. This is a very complicated process for preparing apparently constitutive promoters.
Furthermore, promoters have been described which have specificities for the anthers, ovaries, flowers, leaves, stalks, roots and seeds. The stringency of the specificity and also the expression activity of said promoters is very different. Promoters which may be mentioned are those which ensure leaf-specific expression, such as the potato cytosolic FBPase promoter (WO 97/05900), the rubisco (ribulose-1,5-bisphosphate-carboxylase) SSU (small subunit) promoter, the potato ST-LSI promoter [Stockhaus et al. (1989) EMBO J 8:2445-2245], the mainly leaf-specific ferredoxin NADPH oxidoreductase promoter (FNR promoter) which has a light-inducible element [Oelmüller et al. (1993) Mol. Gen. Genet. 237:261-72] or the leaf-specific promoter of the triose-phosphate translocator (TPT).
Examples of further promoters are promoters with specificity for tubers, storage roots or roots, such as, for example, the patatin class I promoter (B33), the potato cathepsin D inhibitor promoter, the starch synthase (GBSS1) promoter or the sporamin promoter, fruit-specific promoters such as, for example, the tomato fruit-specific promoter (EP-A 409625), fruit maturation-specific promoters such as, for example, the tomato fruit maturation-specific promoter (WO 94/21794), flower-specific promoters such as, for example, the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593).
A promoter regulated as a function of development is described, inter alia, in Baerson et al. (Baerson S R, Lamppa G K (1993) Plant Mol Biol 22(2):255-67).
Promoters have been described which have tissue specificity for the mesophyll and the pallisade cells in leaves (Broglie et al. (1984) Science 234:838-845), the dividing shoot and the root meristem [Atanassova et al. (1992) Plant J 2:291-300], pollen [Guerrero et al. (1990) Mol Gen Genet 224:161-168], seed endosperm [Stalberg et al. (1993) Plant Mol Biol 23:671-6839, root epidermis [Suzuki et al. (1993) Plant Mol Biol 21:109-119], and for the root meristem, root vascular tissue and root knots [Bogusz et al. (1990) Plant Cell 2:633-641].
Other known promoters are those which control expression in seeds and plant embryos. Examples of seed-specific promoters are the phaseolin promoter [U.S. Pat. No. 5,504,200, Bustos M M et al. (1989) Plant Cell 1(9):839-53], the promoter of the 2S albumin gene [Joseffson L G et al. (1987) J Biol Chem 262:12196-12201], the legumin promoter [Shirsat A et al. (1989) Mol Gen Genet 215(2):326-331], the promoters of the USP [unknown seed protein; Bäumlein H et al. (1991) Molecular & General Genetics 225(3):459-67], of the napin gene [Stalberg K, et al. (1996) Planta 199:515-519], of the saccharose-binding protein (WO 00/26388) and the LeB4 promoter [Bäumlein H et al.(1991) Mol Gen Genet 225:121-128]. Said promoters control seed-specific expression of storage proteins.
Many of the abovementioned promoters exhibit, in addition to the primary activity, also “secondary activities” in other tissues.
Owing to the tissue-dependent expression pattern, the abovementioned tissue-specific promoters are poorly suited to expressing selection markers. Here, a selection in all tissue parts, if possible, is required in order to ensure efficient selection.
The “constitutive” promoters described in the prior art have one or more of the following disadvantages:    1. Inadequate homogeneity of expression:            The known “constitutive” promoters frequently display a different level of expression, depending on the type of tissue or cell. Moreover, the expression property is often highly dependent on the site of insertion into the host genome. This indicates that the effects to be obtained by heterologous expression cannot be achieved to the same extent homogeneously in the plant. Under or over dosages may occur. This may have an adverse effect on plant growth or plant value.            2. Inadequate time profile:            The “constitutive” promoters known in the prior art often exhibit a nonconsistent activity during the development of a tissue. As a result, it is not possible, for example, to achieve desirable effects (such as selection) in the early phase of somatic embryogenesis which would be advantageous, especially here, due to the sensitivity of the embryo to in vitro conditions and stress factors.            3. Inadequate applicability to many plant species:            The “constitutive” promoters described in the prior art are often not active in the same way in all species.            4. Gene silencing            If a plurality of expression cassettes with in each case the same “constitutive” promoter are present in an organism, interactions between said expression cassettes and even switching-off (gene silencing) of individual expression cassettes may occur (Mette et al. (1999) EMBO J. 18:241-248).            5. Viral and bacterial promoters            Promoters of viral origin may be influenced by virus infections of the transgenic plant and may then no longer express the desired property (Al-Kaff et al. (2000) Natur Biotechnology 18:995-99).        The public acceptance toward the use of promoters and elements from plant systems is higher than for viral systems.        The number of promoters suitable for expressing selection markers in plants is low and said promoters are usually of viral or bacterial origin.        
An ideal constitutive promoter should have as many of the following properties as possible:    a) A gene expression which is as homogeneous as possible with regard to location and time, i.e. an expression in as many cell types or tissues of an organism as possible during the various phases of the developmental cycle. Desirable expression should take place as early as in the embroyo stage. Furthermore, an efficient selection in dedifferentiated cells (various callus phases) from a tissue culture and other developmental stages suitable for tissue culture is desired.    b) An applicability to various plant species, which is as broad as possible, and applicability to species in which it is not possible to achieve any expression using the “constitutive” promoters known to date.    c) In order to combine a plurality of transgenes in one plant, it is desirable to carry out a plurality of transformations in succession or to use constructs with a plurality of promoter cassettes, but without generating silencing effects due to the multiple use of identical regulatory sequences.    d) A plant origin in order to avoid problems of acceptance by the consumer and possible problems of future approval.